Podcast
Questions and Answers
What is Real-Time PCR?
What is Real-Time PCR?
- A technique for visualizing DNA sequences without amplification
- A process for amplifying target DNA using conventional PCR techniques
- A method for determining the presence of target DNA after the PCR reaction
- A modification of conventional PCR where product accumulation is quantified in real time (correct)
What is the main difference between Real-Time PCR and conventional PCR?
What is the main difference between Real-Time PCR and conventional PCR?
- Real-Time PCR does not require a thermal cycler, unlike conventional PCR
- Conventional PCR can quantify product accumulation 'in real time'
- Conventional PCR is faster than Real-Time PCR
- Real-Time PCR allows for quantification of product accumulation during the reaction, while conventional PCR does not (correct)
What can be determined using Real-Time or Quantitative (qPCR)?
What can be determined using Real-Time or Quantitative (qPCR)?
- The size of the DNA sample used in the PCR reaction
- The temperature at which the PCR reaction occurs
- The type of DNA polymerase used in the PCR reaction
- The amount of target sequence or gene present in a sample (correct)
What is the mechanism behind SYBR® Green detection in qPCR?
What is the mechanism behind SYBR® Green detection in qPCR?
What is the function of TaqMan® probe in qPCR?
What is the function of TaqMan® probe in qPCR?
What happens to the TaqMan® probe during qPCR?
What happens to the TaqMan® probe during qPCR?
What is the Cycle Threshold (Ct) in qPCR?
What is the Cycle Threshold (Ct) in qPCR?
What does the Threshold line represent in qPCR?
What does the Threshold line represent in qPCR?
What does the Baseline (negative control) represent in qPCR?
What does the Baseline (negative control) represent in qPCR?
What does a low Ct value in qPCR indicate?
What does a low Ct value in qPCR indicate?
What is typically expected in samples with high Ct values in qPCR?
What is typically expected in samples with high Ct values in qPCR?
In qPCR, what is the general relationship between Ct values and target sequence abundance?
In qPCR, what is the general relationship between Ct values and target sequence abundance?
Quel est le rôle d'un gène de référence dans la qPCR?
Quel est le rôle d'un gène de référence dans la qPCR?
Pourquoi est-il important que le gène de référence soit présent à une concentration constante dans tous les échantillons?
Pourquoi est-il important que le gène de référence soit présent à une concentration constante dans tous les échantillons?
Quel est le rôle de la sonde TaqMan® dans la qPCR?
Quel est le rôle de la sonde TaqMan® dans la qPCR?
What is required for each reaction in Absolute Quantitation analysis?
What is required for each reaction in Absolute Quantitation analysis?
What is used to quantitate the concentration of unknown experimental samples in Absolute Quantitation analysis?
What is used to quantitate the concentration of unknown experimental samples in Absolute Quantitation analysis?
What is often identified using Absolute Quantitation analysis in qPCR?
What is often identified using Absolute Quantitation analysis in qPCR?
What method is used to express the ratio between the Reference Gene and the Target Gene in qPCR?
What method is used to express the ratio between the Reference Gene and the Target Gene in qPCR?
Why is it crucial for the Reference Gene to remain unchanged in relative quantitation analysis?
Why is it crucial for the Reference Gene to remain unchanged in relative quantitation analysis?
What does the accuracy of relative quantitation depend on in qPCR?
What does the accuracy of relative quantitation depend on in qPCR?
What is one of the uses of qPCR in Pharmaceutical Biotechnology Quality Control in Biopharmaceutical Manufacturing?
What is one of the uses of qPCR in Pharmaceutical Biotechnology Quality Control in Biopharmaceutical Manufacturing?
What does qPCR help to ensure in Viral Safety Testing of biopharmaceutical products?
What does qPCR help to ensure in Viral Safety Testing of biopharmaceutical products?
In what way does qPCR contribute to Toxicology Studies of pharmaceuticals?
In what way does qPCR contribute to Toxicology Studies of pharmaceuticals?
What is reverse transcription PCR (RT-PCR) used for?
What is reverse transcription PCR (RT-PCR) used for?
What is the function of complementary DNA (cDNA) in RT-PCR?
What is the function of complementary DNA (cDNA) in RT-PCR?
Which enzyme is used to synthesize cDNA in RT-PCR?
Which enzyme is used to synthesize cDNA in RT-PCR?
What is necessary to start the experiment with in order to clone the human gene for the insulin protein?
What is necessary to start the experiment with in order to clone the human gene for the insulin protein?
What is the specific starting material required for cloning the human gene for the insulin protein?
What is the specific starting material required for cloning the human gene for the insulin protein?
Which type of cells should actively express the insulin gene for cloning the human gene for the insulin protein?
Which type of cells should actively express the insulin gene for cloning the human gene for the insulin protein?
What is the purpose of Oligo d(T) Affinity chromatography column?
What is the purpose of Oligo d(T) Affinity chromatography column?
Why is RNA rapidly degraded in the cell?
Why is RNA rapidly degraded in the cell?
What type of RNA does the total RNA fraction obtained after cell lysis contain?
What type of RNA does the total RNA fraction obtained after cell lysis contain?
What is an important consideration for the quality of RNA in quantification?
What is an important consideration for the quality of RNA in quantification?
How should the RNA sample be checked for quality?
How should the RNA sample be checked for quality?
Why is it important to ensure the RNA sample is free of genomic DNA (gDNA) contamination?
Why is it important to ensure the RNA sample is free of genomic DNA (gDNA) contamination?
What is the function of oligo (dT) primers in reverse transcription?
What is the function of oligo (dT) primers in reverse transcription?
What is the role of random primers in reverse transcription?
What is the role of random primers in reverse transcription?
How long is the typical incubation period for the enzymatic synthesis of cDNA from RNA template in reverse transcription?
How long is the typical incubation period for the enzymatic synthesis of cDNA from RNA template in reverse transcription?
Flashcards
Real-Time PCR (qPCR)
Real-Time PCR (qPCR)
A lab technique to amplify and measure specific DNA sequences in real-time.
SYBR Green
SYBR Green
A fluorescent dye binding to double-stranded DNA during PCR.
TaqMan Probe
TaqMan Probe
Labeled oligonucleotide specific to a DNA sequence; degraded during PCR to release fluorescence.
Cycle Threshold (Ct)
Cycle Threshold (Ct)
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Ct Value and Abundance
Ct Value and Abundance
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Reference Gene
Reference Gene
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Absolute Quantitation
Absolute Quantitation
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Relative Quantitation
Relative Quantitation
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Reverse Transcription PCR (RT-PCR)
Reverse Transcription PCR (RT-PCR)
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cDNA
cDNA
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RNA Quality
RNA Quality
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Oligo d(T) primers
Oligo d(T) primers
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Cloning Human Insulin
Cloning Human Insulin
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Pharmaceutical Biotechnology applications
Pharmaceutical Biotechnology applications
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Fluorescence
Fluorescence
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Baseline
Baseline
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Background signal
Background signal
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Pharmaceutical Analysis
Pharmaceutical Analysis
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Contaminants
Contaminants
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Quality Control
Quality Control
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Study Notes
Real-Time PCR (qPCR)
- Real-Time PCR (qPCR) is a laboratory technique used to amplify and quantify specific DNA sequences in real-time.
- The main difference between Real-Time PCR and conventional PCR is the ability to quantify the amplified DNA in real-time.
Applications of Real-Time PCR
- Real-Time PCR can be used to determine the presence or absence of a specific DNA sequence, quantify the amount of a specific DNA sequence, and detect genetic variations.
- Real-Time PCR can also be used to analyze gene expression, detect pathogens, and identify genetic mutations.
Mechanism of SYBR Green Detection
- SYBR Green is a fluorescent dye that binds to double-stranded DNA.
- As the polymerase synthesizes new DNA strands, SYBR Green binds to the amplified DNA, and the fluorescence emitted is proportional to the amount of amplified DNA.
TaqMan Probe
- A TaqMan probe is a fluorescently labeled oligonucleotide that is specific to the target DNA sequence.
- The TaqMan probe is degraded by the 5' nuclease activity of the Taq polymerase during PCR, releasing the fluorescent dye.
- The amount of fluorescence emitted is proportional to the amount of amplified DNA.
Cycle Threshold (Ct) and Baseline
- The Cycle Threshold (Ct) is the cycle at which the fluorescence signal is detectable above the background signal.
- The Threshold line represents the level of fluorescence that is considered significant.
- The Baseline (negative control) represents the fluorescence signal in the absence of the target DNA sequence.
Ct Value and Target Sequence Abundance
- A low Ct value indicates a high abundance of the target DNA sequence.
- A high Ct value indicates a low abundance of the target DNA sequence.
Reference Gene
- A reference gene is a gene that is present at a constant concentration in all samples.
- The reference gene is used as an internal control to normalize the expression of the target gene.
Absolute Quantitation Analysis
- Absolute Quantitation analysis requires a standard curve to relate the Ct value to the concentration of the target DNA sequence.
- The concentration of unknown samples is determined by comparing their Ct values to the standard curve.
Relative Quantitation Analysis
- Relative Quantitation analysis compares the expression of the target gene to that of a reference gene.
- The ratio of the target gene to the reference gene is calculated using the 2^(-ΔΔCt) method.
Pharmaceutical Biotechnology Applications
- qPCR is used in Pharmaceutical Biotechnology Quality Control to detect and quantify contaminants in biopharmaceutical products.
- qPCR helps ensure the safety of biopharmaceutical products by detecting viral contaminants.
- qPCR is used in Toxicology Studies to detect and quantify gene expression in response to pharmaceuticals.
Reverse Transcription PCR (RT-PCR)
- Reverse Transcription PCR (RT-PCR) is used to amplify RNA sequences.
- Complementary DNA (cDNA) is synthesized from RNA using reverse transcriptase.
- cDNA is used as a template for PCR amplification.
Cloning the Human Insulin Gene
- The human insulin gene can be cloned using mRNA isolated from cells that actively express the insulin gene.
- Oligo d(T) Affinity chromatography is used to isolate mRNA from total RNA.
- RNA is rapidly degraded in the cell, so it is essential to work quickly and carefully to preserve the RNA.
RNA Quality and Primers
- RNA quality is critical for successful reverse transcription and PCR amplification.
- RNA should be checked for quality using agarose gel electrophoresis or bioanalyzer.
- Oligo d(T) primers and random primers are used in reverse transcription to synthesize cDNA.
- It is essential to ensure the RNA sample is free of genomic DNA (gDNA) contamination.
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Description
Test your knowledge of Real-Time PCR, a modification of conventional PCR used to quantify and visualize product accumulation in real time as the reaction progresses.
